Rev. sci. tech. Off. int. Epiz., 1991, 10 (2), 393-408 Viral haemorrhagic disease of rabbits in the People's Republic of China: epidemiology and virus characterisation WEI-YAN XU * Summary: Viral haemorrhagic disease (VHD) is a new and severe infectious disease of rabbits, with a high rate of morbidity and mortality. The disease occurs throughout the year, affecting only adult rabbits and not other domestic animals, fowls or laboratory rodents. The transmission is horizontal, by direct or indirect contact, and through all routes. There is no evidence of congenital infection or biological vectors. The causative agent, viral haemorrhagic disease virus (VHDV), is present in all tissues, excretions and secretions. It is an icosahedral and nonenveloped parvo-like virus. The genome, as determined by classical methods, high performance chromatography and in vitro synthesis of double-stranded DNA, is linear, single-stranded DNA. VHDV can agglutinate human erythrocytes at very high titres, irrespective of blood groups, and has a stable reaction to many physical and chemical factors. VHDV has been adapted to grow on rabbit kidney cell strain (DJRK) culture and to produce cytopathic effect (CPE). Inactivated cell culture can protect inoculated rabbits against virulent VHDV. The disease is now effectively controlled in the People's Republic of China, but has not yet been completely eradicated. KEYWORDS: Cell culture - Epidemiology - Genome cloning Haemagglutination - Haemorrhagic disease - High performance chromatography - Parvovirus - Single-stranded DNA - Transmission - Viral haemorrhagic disease virus of rabbits. INTRODUCTION Viral haemorrhagic disease (VHD) is a new, peracute and highly communicable disease, affecting adult rabbits only. It is characterised by sudden death, haemorrhages in various tissues, bloody exudates in the trachea and a r o u n d the nostrils, and by haemagglutination (HA) of h u m a n red blood cells at a very high titre. The aetiological agent is a unique virus which was not reported in worldwide literature before 1984. Owing to contradictory results reported on the nature of the genome, n o consensus has been reached concerning its classification as a virus. The author bases his assumption that it is a parvo-like virus on the type of genomic nucleic acid involved and on cross-reaction with certain other parvoviruses. * Department of Veterinary Science, Nanjing Agricultural University, 210014 Nanjing, Jiangsu Province, People's Republic of China. 394 C H A R A C T E R I S A T I O N OF VIRAL HAEMORRHAGIC DISEASE VIRUS Morphology The morphology of viral haemorrhagic disease virus (VHDV) was examined under the electron microscope by negative staining with phosphotungstic acid after a sample from the liver emulsion of an infected rabbit had been concentrated and purified by differential centrifugation and polyethylene glycol (PEG) sedimentation, or further by extraction with chloroform and other organic solvents. V H D V is approximately 32-34 nm in diameter and varies slightly in different preparations (2, 3, 4, 6, 9, 13, 16, 18, 20, 26, 29). The virion is icosahedral a n d nonenveloped a n d has a core of 18-20 nm (4, 9, 13). The capsid is composed of 32 capsomeres (9, 18) (Fig. 1). The virions in thin sections of rabbit liver and kidney cell strain (DJRK) from infected cell culture are slightly smaller and are found in the cell nuclei and around the nuclear m e m b r a n e (8, 14, 17). FIG. 1 Viral haemorrhagic disease virus, negative staining, x 216,000 Sedimentation coefficient and buoyant density The purified virus preparation is layered on a continuous sucrose gradient from 20 to 6 0 % and centrifuged at 18,400 r p m . The sedimentation coefficient of V H D V , determined by ultraviolet scanning, is 162S. The purified virus preparation layered 395 on 3 5 % CsCl is centrifuged at 40,000 r p m for 24 h. The virus band is collected and examined with A b b é ' s refractometer. The buoyant density of V H D V is 1.36-1.38 g / c m (2, 10). 3 Nucleic acid Because the virus is difficult to grow on cell culture and to purify without contamination of host cell components, there was much disagreement among Chinese scientists between 1984 and 1986 over the nature of the nucleic acid of V H D V . The dispute subsided after highly purified preparations and modern techniques, complementing classical methods, had been used. Type The partially purified virus preparation is further treated with ribonuclease (RNase) and deoxyribonuclease (DNase) to degrade the contaminating nucleic acids of host cells adsorbed on the virion capsid. The highly purified virus preparation is examined by diphenylamine and orcinol reactions and the nuclease degradation test (2, 18, 20). The electrophoretically pure nucleic acid is also analysed by high performance liquid chromatography for its base composition (20, 21). The results are summarised in Table I and Figure 2. TABLE I Identification of the type of nucleic acid of VHDV Procedure VHDV-NA Yeast RNA Thymus DNA Diphenyl amine Orcinol DNase 1 RNase A Base composition Blue No change Degraded Resistant No change Dark green Resistant Degraded Blue Light green Degraded Resistant C, G, A , T C, G, A , U C, G, A, T Since V H D V nucleic acid turns blue in the diphenylamine reaction and light green in the orcinol reaction, is degraded by DNase 1 but resistant to RNase A , and is composed of four nucleoside bases, adenine (A), thymine (T), guanine (G) and cytosine (C), there is n o doubt that it is a D N A . Strand The highly purified virus preparation has been subjected to several tests. In the melting point (Tm) test, the purified nucleic acid is dissolved in standard saline-citrate buffer (0.15 M NaCl and 0.015 M sodium citrate, p H 7.0). The optical density (OD) values of the solution are recorded every 5 min from 18 C to 95 °C by ultraviolet spectrometry at a wavelength of 260 n m . The O D values increase only slightly from 0.25 to 0.28. This indicates that the V H D V nucleic acid cannot be separated by heat, unlike that of the double-stranded (ds) D N A . In acridine orange staining the nucleic acid band in agarose gel, after electrophoresis, gave a red flame when examined under a high pressure mercury l a m p . In the nuclease degradation test, the nucleic acid was degraded by Alu 1, but resistant to DNase S1 (2, 20). In high performance liquid 2 Left: VHDV nucleic acid Middle: calf thumus DNA Right: yeast RNA Note the difference in the four nucleotide bases between DNA and RNA C = cytosine G = guanine T = thymine A = adenine U = uridine High performance liquid chromatogram of hydrolysates of VHDV nucleic acid FIG. 396 397 chromatography, A = 2 4 . 4 % , T = 3 1 . 9 % , C = 2 3 . 7 % and G = 2 0 . 8 % (19). Since G is paired with C, and T with A , the mole percentages of G and C, or T and A , have to be approximately equal in d s D N A . Here G and C, and T and A , are unequal, which provides indirect evidence that the nucleic acid tested is single-stranded. T h e results are summarised in Table II. TABLE II Identification of the nature of the nucleic acid strand of Nucleic acid of VHDV Procedure Control Phage X ssDNA Calf thymus dsDNA OD value increase at 260 nm Green Resistant Degraded Heat denaturation No change No change Acridine orange DNase SI Alu 1 Base composition by HPLC Red Degraded Resistant Red Degraded Resistant C G T A = = = = 23.7 20.8 31.9 24.4 VHDV C G T A Not done = = = = 21.2 21.5 27.8 28.2 Shape After elimination of the contaminating cellular nucleic acid with nucleases, the purified virions are treated with urea to elicit the release of the nucleic acid from the capsid. After being spread on water, stained with uranium acetate and shadowed with platinum, the specimen is observed under the electron microscope. As shown in Figure 3, the nucleic acid is not segmented and is linear in shape, with an average length of 1.92 µm (1.84-2.2 µm) (28). The results of Polyacrylamide gel electrophoresis (PAGE) and agarose gel (AGE) also indicate that the nucleic acid of V H D V is not segmented (2). Molecular weight 6 The molecular weight of V H D V nucleic acid is 2.4 x 10 when compared with the distance moved by phage M 13 single-stranded D N A after P A G E and A G E . The molecular weight calculated from the length of nucleic acid, by multiplying by 1.2 x 10 per µm, is 2.3 X 10 . These two values are very close. 6 Terminal 6 hairpin The evidence of classical tests and high performance chromatography indicates that the genome of V H D V is single-stranded D N A . A m o n g vertebrate viruses, only the genome of the Parvoviridae belongs to this type. If V H D V is a parvovirus, there should be hairpin structures at the 3 ' - and 5'-terminals of the nucleic acid chain. To examine this, the V H D V nucleic acid is extracted from the highly purified viral preparation and reacted with Escherichia coli D N A polymerase 1 Klenow large 398 FIG. 3 Electron micrograph of D N A of viral haemorrhagic disease virus, x 20,000 fragment. Double-stranded D N A is synthesised in vitro and then integrated into the Bluescript plasmid. The recombinant plasmids collected from transformed E. coli are identified by agarose electrophoresis and restriction enzyme cleavage. They could hybridise with V H D V nucleic acid by dot blot hybridisation after being labelled by nick translation (24). The evidence indicates that there is a hairpin at the 3'-terminal of V H D V nucleic acid. Polypeptides T h e emulsion of infected liver was purified by a process of repeated freezing and thawing, chloroform extraction, P E G sedimentation and differential centrifugation. The partially purified V H D V preparation was chromatographed on a Sepharose 4B column. Three fractions were eluted. The purified and concentrated V H D V was in fraction two when identified by H A and A G E . This fraction was analysed for viral polypeptides with sodium dodecyl sulphate (SDS)-PAGE. Four polypeptides were detected; their respective molecular weights, as compared with those of marker proteins, were as follows: VP1 = 58.3-61.1 kDa, V P 2 = 54.7 kDa, VP3 = 51-53 kDa and VP4 = 25.9 kDa. When the electrophoretic gel was scanned with a densitometer, the average component percentages, calculated from the 4-test integral area, were: VP1 = 54.7 ± 1.2%, V P 2 = 17.7 ± 0 . 6 % , VP3 = 22.7 ± 1.1% and V P 4 = 4.9 ± 0 . 1 % (7, 11). In Western blotting, the polypeptides were transferred from the electrophoretic gel onto nitrocellulose filters and reacted with: a) V H D antiserum and peroxidase-labelled staphylococcal protein A , or 399 b) monoclonal antibodies from several hybridoma cell strains and peroxidaselabelled antimouse IgG goat serum. All four polypeptides were detected by V H D antiserum and three monoclonal antibodies (25, 27) (Table III). It is suggested that polypeptides V P 1 , V P 2 , V P 3 and VP4 share c o m m o n epitopes, similar to those found in Aleutian mink disease virus. TABLE III Immunological reactions VHD V specific monoclonal antibodies and VHD V according to Western blotting between Name of MAb VHDV polypeptides VP2 VP3 VP1 AA8-1 CF2-1 CG4-1 CH3-1 RM-14 RM-17 + + + - - - + + •+ + - polypeptides, VP 4 + - + + + + - • - - - - - Haemagglutination Spectrum Various tissues collected from infected rabbits immediately before and after death can agglutinate h u m a n red blood cells at very high titres. N o obvious difference is observed among blood groups A, B, A B and O. Erythrocytes of the chicken, goose and sheep seem to be agglutinated at low titres (10 x 2 - 10 X 2 ), while those of other animals, including the cow, goat, pig, rabbit, rat, mouse, guinea pig, duck and quail, cannot be agglutinated. H A is inhibited by specific antiserum. 2 4 Distribution The virus exists in all tissues and body fluids of infected animals. The H A titres in the liver, spleen and serum can reach 1 0 x 2 - 1 0 x 2 , much higher than those of other tissues (Fig. 4). There is some evidence that the H A titre is related to the content of blood in the tissue. 1 0 Physical and chemical 1 8 factors H A is not significantly affected at different temperatures (4°C-37°C). There is no strict p H value requirement; the H A titre, optimal at p H 6.0, is basically the same between p H 4 and 7.2. When the agglutinated red blood cells (RBC) are suspended in borate buffer at p H 9.2 and incubated at 22°C-37°C for 30 min, the viral particles are released. The released RBC and virus are able to reagglutinate when the p H value lowers. T w o percent NaCl in 0.01 M phosphate buffer is usually used as a diluent of RBC in the H A test; this can enhance the H A titre to a value 1-2 times higher than in 0 . 8 5 % NaCl (15, 23). HA titre n (10x2 ) 400 FIG. 4 H A titres of various infected tissues Li = liver S = spleen H = heart M = muscle Br= brain Bl = blood Lu=tung K = kidney Ly= lymph node The H A activity can be destroyed by 0 . 5 % trypsin, 2 % sodium borohydride, or 2 % chloramine T, but not by receptor destroying enzyme (RDE), sodium metabisulphite, potassium periodate, chloroform, ether or formaldehyde. The H A activity is rather stable to heat. When the infected tissue is kept at 4 ° C and 15°C for three m o n t h s , the H A titres decrease by 2 - 2 and 2 - 2 , respectively. If it is kept in a waterbath at 56°C for 24 h, the titres decrease by 2 - 2 (23). 2 4 4 5 4 Receptors 6 on RBC The viral haemagglutinin receptors on the surface of h u m a n RBC are sensitive to chloroform and ether, and stable to trypsin, potassium periodate, R D E an 401 sodium borohydride. They are highly resistant to heat, unaffected after heating at 95°C for 15 min. The RBC fixed with 1% K M n 0 remain agglutinable by the virus at 4 ° C , with a clear and stable agglutination pattern. Comparative study indicates that H A results with either fixed or fresh RBC correlate well (22). 4 Resistance The 10% liver emulsion of infected rabbits was centrifuged at 10,000 x g for 30 min. The supernatant was collected and treated in each of the following ways: a) in a waterbath at 50°C for 60 min; b) in 1.0 M M g C l solution in a waterbath at 50°C for 60 min; 2 c) in p H 3.0 H a n k s ' solution at r o o m temperature for 30 min; d) in 2 0 % ether stored overnight at 4 ° C ; e) in 10% chloroform, vibrating vigorously at r o o m temperature for 10 min; f) control, without any treatment. Samples of each preparation were inoculated peritoneally into six or seven susceptible rabbits. Rabbits of the six groups all became infected and died within 36 h; however, one rabbit in G r o u p 3 (preparation c) survived, as did another in Group 4 (preparation d) (4). Alternately, the supernatant of the liver emulsion was mixed with an equal volume of 2 % N a O H or 2 % formalin and was incubated at 37°C for 1 h. Each mixture was diluted tenfold by phosphate buffered saline (PBS) and inoculated into six susceptible rabbits, all of which survived and remained healthy, while the control rabbits inoculated with untreated sample died within 36 h (20). The virus is very stable at low temperatures. Infected liver samples kept at - 5°C for 413 days or - 2 0 ° C for 560 days did not have significant drops in their infective titres. Those kept at - 7 0 ° C for 4.5 years after lyophilisation remained infectious without any loss (16). F o r safety purposes rabbit sheds, including the cages, floors, walls and utensils, should be cleaned and thoroughly disinfected with 10% bleaching powder or 3 % formalin followed by 2 % N a O H , and kept empty for two weeks in summer, or for two m o n t h s in colder seasons. Relationship to some parvoviruses In indirect E L I S A , when purified V H D V is coated on polystyrene microtitration plate wells and treated with V H D V reference antiserum and peroxidase-conjugated staphylococcal protein A , or with V H D V monoclonal antibodies and peroxidaseconjugated antimouse IgG goat serum, the titre of V H D V is 1:6400. The results have been compared with those of the canine (CPV), feline (FPV), mink (MPV), pig (PPV), goose (GPV) and mouse (MVM) parvoviruses. When VHDV-antiserum is used, the ELISA titres of these viruses are 1:160, 1:80,1:40, 1:80, 1:160 and 1:160, respectively. When purified G P V is coated on microtitration plate wells and reacted with antiserum to G P V or V H D V , the respective titres are 1:12800 and 1:320. When purified M V M is used as antigen and reacted with antisera to M V M and V H D V , the titres are 1:9600 and 1:640, respectively. If the coated antigen is P P V and is reacted with antisera to P P V and V H D V , the respective titres are 1:12800 and 1:320. If the coated antigen 402 is M E V and is reacted with antisera to M E V and V H D V , the titres are, respectively, 1:5120 and 1:40. The results indicate that V H D V may, to some extent, be related in antigenicity to M V M , P P V and G P V . Cell culture VHDV is very difficult to grow in cell culture. Since 1984, the author has inoculated emulsions of liver, spleen, lung and serum collected from experimentally infected rabbits onto various cell cultures, including primary rabbit cells (kidney, liver, lung and testis) and cell lines (PK-15, BHK-21, MA-104, IBRS-2, HeLa and VERO). Blind passages were made and every passage was checked for H A and cytopathic effect (CPE). None of them manifested any evidence of growth. The author also modified the method of inoculation; for example, by inoculating just after adsorption of cells on a glass surface, inoculating at phase SI of cell division, treating the sample with trypsin and adding trypsin to the cell medium, substituting rabbit serum for calf serum, and using cells together with culture fluid in passage. None of the attempts succeeded. In 1988, a rabbit kidney cell strain, designated DJRK, was used. Regular C P E was observed at the third passage; this was characterised by the rounding and aggregating of cells which finally detached from the glass surface (Fig. 5). Thread-like intercellular bridges were also observed (8). Fluorescence, principally in the cytoplasm around the cell nuclei and, to some extent, in the nuclei at an early stage, was detected by the fluorescent antibody technique. The cell cultures of the fifth and tenth passages were inoculated into susceptible rabbits, all of which died within 5 to 11 days. H A appeared to titres of 2 - 2 at 1-3 days after inoculation, 2 - 1 2 at 2-8 days after inoculation and increased abruptly to 2 - 2 just before death. The pathological changes were similar to the natural VHDV infection. Numerous virions accumulated adjacent to the nuclei and a few were observed in the nuclei themselves, in thin sections of cultured cells of the tenth passage (Fig. 6). Susceptible rabbits, inoculated with cell culture of the tenth or fifteenth passages inactivated by 0.35% formaldehyde, could produce HI serum antibodies to titres of 2 -211 at day 10 post inoculation and could resist the challenge of virulent V H D V at day 15 post inoculation. 2 6 6 14 12 17 9 FIG. 5 Cytopathic effect (CPE) produced in a monolayer of DJRK cell culture by VHDV Rounding of cells and intercellular bridges can be observed ____ • _ 12126 - 1 7 | y y 9K» X46K FIG. 6 Thin section of a monolayer of DJRK cells infected by VHDV, x 80,000 Virions aggregate adjacent to the disappearing nuclear membrane; a few are seen in the nucleus 404 EPIDEMIOLOGY Host range It is probable that only rabbits are susceptible to V H D . N o evidence indicates that horses, mules, donkeys, cattle, buffalo, sheep, goats, swine, dogs, cats or fowls can be affected by the disease, though these animals have frequent contact with infected rabbits and rabbit carcasses, excretions and virus-contaminated materials in backyard rabbitries (5, 13, 16). M a n is not susceptible to V H D V , as evidenced by the lack of disease in the millions of people who consume rabbit meat. N o case of infection has been reported from large numbers of farmers, veterinarians and laboratory technicians who come in contact with V H D V daily. Attempts to infect laboratory rodents and fowls have all been unsuccessful. It has been reported that white mice, rats (white, black, grey and brown), golden hamsters and guinea pigs have been inoculated subcutaneously, intramuscularly, peritoneally or orally with liver emulsions of infected rabbits. N o symptom has manifested in these animals and H I antibodies cannot be detected in their sera (16). A captive hare with pathological changes similar to V H D was found in 1986. Its liver emulsion was inoculated into rabbits and typical V H D resulted. The H A titre of that liver sample was 10 x 2 , which could be inhibited by V H D V antiserum. Ninety-nine serum samples taken from hares had titres lower than l:21-l:2 (40 hares), 1:2 and 1:2 -1:2 (25 hares), and 1:2 -1:2 (9 hares). Hunters have never seen any hares either infected or killed by a disease resembling V H D . 6 3 4 5 7 8 9 Susceptibility of rabbits Domestic rabbits of all breeds appear to be fully susceptible. W h e n A n g o r a , Rex, Chinchilla, Japanese White and fur hybrids are inoculated experimentally with virus preparation, n o significant difference in morbidity and mortality is observed. In natural outbreaks, however, wool rabbits may be more susceptible t h a n meat and fur rabbits (16). Susceptibility is clearly influenced by age. Rabbits less t h a n one m o n t h of age cannot be infected clinically, and those between one and two months of age may be infected at low percentages. The majority of infections are found in rabbits older t h a n three m o n t h s . There is n o difference in susceptibility between male and female adult rabbits (13). Serum HI antibodies may exert effects on susceptibility. W h e n the antibody level is above 2 , rabbits are usually resistant to experimental challenge (12). Forty-six samples of rabbit serum collected in Jiangsu Province from 1975 to 1983 were examined for H I antibodies to V H D . Titres of 44 samples were below 2 ; those of only two samples reached 2 . This implies that V H D did not exist in Jiangsu province before 1984, but the source of antibodies is not clear. Naturally resistant rabbit flocks are not uncommon, even in isolated villages where V H D never occurs and vaccination is not carried out (1). 4 2 4 Transmission The V H D virus is present in blood, organs, secretions, excretions and on skin and mucous membranes, principally during the late stage of infection and after death. 405 Direct or indirect contact with infected rabbits or carcasses, excretions and contaminated objects m a y transmit infection to susceptible animals. Rabbit wool pedlars, who shear and purchase rabbit wool even from infected rabbits and carcasses, may play a significant role in the spread of the disease in the People's Republic of China. They come to villages by coaches and trains and the virus can be carried extensive distances on their h a n d s , clothes, shoes and scissors. Through ignorance, farmers may carry the virus to their neighbours and relatives (19). Infected rabbits m a y accidentally be mixed with healthy ones in rabbit processing plants, especially during the incubation period. The virus is difficult to detect in meat; however, by using E L I S A , the author has detected it in bone m a r r o w after viral antigens h a d been concentrated from samples (19). Since 1986, new outbreaks have occasionally occurred in healthy rabbitries after the introduction of breeding rabbits from enzootic areas where vaccination with inactivated vaccine had been regularly carried out. Young rabbits and vaccinated adults may appear to be healthy when, in fact, they are subclinically infected (19). Season V H D is epizootic t h r o u g h o u t the year, without marked seasonal differences, though outbreaks are comparatively more frequent during colder m o n t h s . P R E S E N T S T A T U S OF V I R A L H A E M O R R H A G I C D I S E A S E OF RABBITS IN T H E PEOPLE'S REPUBLIC OF C H I N A The first case of viral haemorrhagic disease, involving an Angora rabbit imported a few days before from the G e r m a n Democratic Republic, appeared in the spring of 1984 in Wuxi City, Jiangsu Province, about 100 km from Shanghai. Except for the young and suckling animals, almost all the rabbits in the affected rabbitry h a d died of infection within a week. Pathogenic bacteria were not isolated and antibiotics were ineffective; no infectious disease with similar symptoms and pathological changes could be found by referring to the worldwide literature. In less than nine m o n t h s , the disease h a d spread rapidly to an area of approximately 50,000 k m , nearly destroying most of the rabbitries within that area. At first, local scientists felt helpless to control this horrible disease; before long, however, an inactivated vaccine was developed which induced solid and effective immunity in rabbits. By the end of 1986 the disease h a d gradually been brought under control, and there is now hope of eradication within a few years. However, the eradication p r o g r a m m e is not always carried out as conscientiously as required by the veterinary authorities. Sporadic outbreaks, usually limited to a single rabbitry or a small village, are still found in other provinces, where vaccinations are not performed with the necessary vigour. The disease readily subsides after all the rabbits of infected rabbitries are slaughtered and the rabbits of the neighbouring rabbitries are vaccinated. The source of infection probably remains rabbits introduced for breeding purposes, even if they appear to be normal. These rabbits are not usually infected in an outbreak because they have been vaccinated, and high levels of H I antibodies are detected in their sera. 2 406 LA MALADIE HÉMORRAGIQUE VIRALE DU LAPIN EN RÉPUBLIQUE POPULAIRE DE CHINE : ÉPIDÉMIOLOGIE ET CARACTÉRISATION DU VIRUS. - Wei-Yan Xu. Résumé: Récemment reconnue, la maladie hémorragique virale (VHD) est une grave maladie infectieuse des lapins, qui entraîne des taux de morbidité et de mortalité très élevés. Elle peut se déclarer à n'importe quelle période de l'année et n'atteint que les lapins adultes à l'exclusion de tout autre animal domestique, volaille ou rongeur de laboratoire. Elle se transmet par voie horizontale, par contact direct ou indirect, et par toutes les voies. Il n'a pas été rapporté de cas d'infection congénitale ni de transmission par vecteurs vivants. Le virus responsable de la maladie hémorragique virale (VHDV) est présent dans tous les tissus infectés, ainsi que dans les excrétions et sécrétions. Il s'agit d'un virus icosaédrique nonenveloppé, proche des parvovirus. Les méthodes classiques d'analyse du génome, telles que la Chromatographie haute performance et la synthèse in vitro de l'ADN double brin, ont permis de déterminer que le génome du VHDV était un ADN linéaire à un seul brin. L'auteur a observé que le VHDV agglutine à un très haut titre les globules rouges humains, quel que soit le groupe sanguin. Le virus reste stable en présence de nombreux agents physiques et chimiques. Le VHDV a été adapté en culture sur des souches cellulaires de rein de lapin (DJRK) où il entraîne un effet cytopathogène. La culture cellulaire inactivée peut protéger les lapins inoculés contre une souche virulente de VHDV. La maladie est à présent bien contrôlée en République populaire de Chine ; elle n'a cependant pas encore été totalement éradiquée. MOTS-CLÉS : ADN à un brin - Chromatographie haute performance - Clonage du génome - Culture cellulaire - Epidémiologie - Hémagglutination - Maladie hémorragique virale du lapin - Parvovirus - Transmission - Virus de la maladie hémorragique virale du lapin. * * * LA ENFERMEDAD HEMORRÁGICA VIRAL DEL CONEJO EN LA REPÚBLICA POPULAR DE CHINA: EPIDEMIOLOGÍA Y CARACTERIZACIÓN DEL VIRUS. Wei-Yan Xu. Resumen: La enfermedad hemorrágica viral del conejo (VHD) es una enfermedad recientemente identificada que ocasiona una morbilidad y una mortalidad importantes. La enfermedad puede declararse en cualquier período del año; sólo afecta los conejos adultos, sin tocar ningún otro animal, sea éste doméstico o de laboratorio. Se transmite horizontalmente a través de todas las vías de transmisión por contacto directo o indirecto. No existe evidencia de infecciones congenitales ni ocasionadas por medio de vectores biológicos. El virus responsable de la enfermedad hemorrágica viral (VHDV) está presente en todos los tejidos, así como en las excreciones y secreciones. Se trata de un virus semejante a los parvovirus, de forma icosaédrica y sin envoltura. Métodos clásicos de análisis del genoma, como la cromatografía altamente performante o la síntesis in vitro del ADN doble fila, determinaron que el genoma del VHDV es un ADN linear de una fila. El VHDV puede aglutinar eritocitos humanos, alcanzando títulos muy elevados, sin distinción entre los grupos sanguíneos. Presenta una buena resistencia a muchos factores físicos y químicos. El VHDV ha sido adaptado en cultivos celulares obtenidos a partir de cepas de riñon de conejo (DJRK), produciendo un efecto citopatogénico. 407 El cultivo celular inactivado puede proteger los conejos inoculados contra una cepa virulenta de VHDV. Actualmente, la enfermedad está bajo control en la República Popular de China, a pesar de no haberse logrado todavía su total erradicación. 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